Figure EV5. APC2 competes with the donor Ub to bind to the UBE2S^HTH .

• A
  Structure of UBE2C bound to the APC11 RING and APC2 WHB (PDB: 5L9U; Brown et al, 2016), demonstrating that the UBE2C^HTH is not interacting with the APC/C.
• B
  Representative fluorescent scan (left) and its quantification (right) monitoring CycB* ubiquitination by APC/C and either UBE2C WT or K157E. Data were statistically analyzed using an unpaired Welch’s t‐test (*P ≤ 0.05, n = 4 independent, technical replicates). Error bars: SEM. Data normalized to the unmodified substrate band from reactions lacking an E2.
• C
  Quantification of Fig 6E monitoring the polyubiquitination of Ub‐CycB* by a panel of UBE2S^HTH charge‐swap variants. Data were statistically analyzed by one‐way ANOVA (*P ≤ 0.05, ****P ≤ 0.0001, n = 3 independent, technical replicates). Error bars: SEM. Data normalized to the unmodified substrate band from reactions lacking an E2.
• D, E
  Fluorescent scan (D) and its quantification (E) monitoring APC/C‐dependent Ub‐Securin (Ub‐Sec*) ubiquitination utilizing the panel of UBE2S variants (related to Fig 6E). Data were statistically analyzed by one‐way ANOVA (n = 3 independent, technical replicates). Error bars: SEM. Data normalized to the unmodified substrate band from reactions lacking an E2.

Source data are available online for this figure.